Keratinocyte stem cells have an unquestioned role in maintaining the normal structure and function of the epidermis and hair follicles and are important players in inherited and acquired skin diseases. Therefore, identification of genes regulating their numbers and proliferative potential is a critical problem in cutaneous biology. We have taken a novel strategy for gene identification taking advantage of several recent advances made by our laboratory including 1) a particularly sensitive and quantitative in vitro assay for keratinocyte stem cells, 2) discovering that the number and size of those colonies are quantitative multigenic traits, and 3) genetically mapping a locus linked to a high number of stem cells forming the largest colonies. The objective of this proposed research is to identify a gene or gene cluster in a locus we have named Keratinocyte stem cell locus 2 (Ksc2) on mouse chromosome 4. Our hypothesis is that a gene (or genes) in this locus regulates the number of stem cells forming the largest keratinocyte colonies. The focus of Specific Aim 1 is to use additional genetic and statistical tools to narrow the linkage interval of the Ksc2 locus that we have previously identified from approximately 10 cM to approximately 0.5 cM. In Specific Aim 2, we will use a candidate gene approach for stem cell gene identification using database mining and lab-based methods. In Aim 3, we will use complementary gene expression profiling to identify differentially over- or under-expressed genes as well as associated molecular pathways, and to assess regulatory polymorphisms causing differences in gene expression. Using the combined, interactive approaches of these three aims, we expect to identify and characterize a new regulatory gene for the keratinocyte stem cells forming the largest colonies, and hence, the highest proliferative potential. The significance of this project is that the gene or gene cluster in the Ksc2 locus, because it shows linkage with a high number of the largest colonies, may be a surprising and unanticipated target for skin cancer therapy and prevention. The approach we present represents a novel application of powerful genetics methods applied in a completely new way to a completely new phenotype: the number and size of keratinocyte colonies. We previously found that colony number reflected the number of proliferative potential of keratinocyte stem cells. This proposed research will impact our understanding of epithelial stem cell regulation. This project not only has basic science ramifications for understanding hair follicle and epidermal homeostasis, it may have clinical impact for the diagnosis, prevention, and treatment of skin cancer, hyperplastic skin disease, as well as potential conditions of declining stem cell function.